The long term objective and goal of this proposal throughout its tenure has been elucidation of factors controlling sarcoplasmic reticulum function in cardiac and skeletal muscle. Since the initial application, the principle objectives and specific aims have related to: 1) characterization of the mechanism by which the calcium ion is translocated across the phospholipid bilayer and the relationship of this translocation to energy transduction and 2) the role of cyclic nucleotides in control of calcium flux by the sarcoplasmic reticulum. The present application continues these goals with the following specific aims: We propose to characterize non-translocated intermediate calcium compartment and its role in the control of calcium translocation across SR membranes by further studying the coupling of incorporation of calcium into this compartment to translocation and factors which influence it. In addition, structural characteristics of this non-translocated compartment will be studied in native and altered SR fractions using x-ray diffraction and neutron diffraction techniques. We wish to examine those factors which control the mechanism of nucleotide hydrolysis and its role in energy transduction in the SR membrane. We will examine factors which influence the characteristics of the enzyme cycle which is associated with nucleotide hydrolysis and its relation to Ca2+ flux. We propose to examine the presence and unique characteristics of adenylyl cyclase associated with the SR with respect to its modulation by G-proteins, calmodulin and Ca2+, and by hormones. We also propose to further characterize the unique G-protein profile in SR fractions as compared to SL fractions and evaluate what roles G-proteins may be playing in SR function. These experiments will involve the use of varying probes including ADP ribosylation, GTP photoaffinity, and specific antibodies to alpha- subunits. These studies will involve both biochemical and electron microscopic investigation to further localize specific G-proteins in regions of SR. The study as a whole deals with the control of excitation- contraction coupling and factors which influence it with regard to the modulation of SR function. The control of membrane function is critical to function of the cardiac muscle and its response to external stimulus.